The broad goal of this proposal is to understand the strategies used by enzymes in the catalysis of phosphoryl transfer reactions, the most common reaction in biology. We focus on this class of enzymes as a prototype for unraveling fundamental features of enzymatic catalysis. Furthermore, phosphoryl transfer is a key transformation in biology, is often perturbed in cancer and other disease states, and enzymes involved are actual or potential therapeutic targets for numerous diseases. The results will not directly lead to inhibitors and potential therapeutics, but increased understanding of the principles underlying catalysis, beyond their intrinsic fundamental importance, may ultimately help transform our ability to design and manipulate inhibitors as drugs. The research proposed herein is also integrally tied to the evolution of new enzymatic activities, a question of fundamental biological interest, and this research has the potential to aid in the development of strategies to design or better select for enzymes with medicinally or economically desirable activities. This proposal focuses on the alkaline phosphatase (AP) superfamily, aiming to understand the underlying structural and functional reasons why different members of this superfamily preferentially catalyze different reactions. Despite structural homology and indistinguishable bimetallo zinc sites, alkaline phosphatase and the AP-superfamily phosphodiesterase (nucleotide pyrophosphatase/phosphodiesterase or NPP) differ in specificity for the hydrolysis of phosphate monoesters and diesters by >1015 fold. Further, both enzymes catalyze hydrolysis of sulfate esters at a greatly compromised rate but with a substantial rate enhancement. Thus, the question is raised: What features of these enzymes lead to optimization for each cognate reaction? The ability to compare catalysis of these reactions, whose substrates differ in charge and transition state nature, provides a powerful means to distinguish enzymatic features involved in specific catalytic mechanisms. A highly multidisciplinary approach will be used: steady state and pre-steady state kinetic comparisons of cognate and non-cognate substrate reactions with wild type and mutant enzymes to determine rate enhancements and to dissect catalysis; structural comparisons between homologous enzymes to guide and interpret site-directed mutagenesis; X-ray crystallography and extended X-ray absorbance fine spectroscopy (EXAFS) to compare structures of homologous enzymes and to determine the structural consequences of mutations; binding of substrates, inhibitors, and transition state analogs to wild type and mutant enzymes to further compare homologous enzymes and determine the energetic consequences of mutations; and linear free energy relationships and heavy atom isotope effects to obtain information about the reactions' transition states and their active site interactions. PUBLIC HEALTH RELEVANCE: Unraveling the fundamental principles that allow enzymes to distinguish between the reactions they catalyze may provide the knowledge to guide the design of new enzymes that catalyze reactions that are medically beneficial or industrially useful. Further, this understanding may ultimately aid in the development of better drugs that target critical phosphorylation events in disease processes.